Primary Structure of Proteins
The primary structure of peptides and proteins refers to the linear number
and order of the amino acids present. The convention for the designation of
the order of amino acids is that the N-terminal end (i.e. the end bearing the
residue with the free α-amino group) is to the left (and the number 1 amino
acid) and the C-terminal end (i.e. the end with the residue containing a free αcarboxyl group) is to the right.
Secondary Structure in Proteins
The ordered array of amino acids in a protein confer regular conformational
forms upon that protein. These conformations constitute the secondary
structures of a protein. In general proteins fold into two broad classes of
structure termed, globular proteins or fibrous proteins. Globular proteins are
compactly folded and coiled, whereas, fibrous proteins are more filamentous
or elongated. It is the partial double-bond character of the peptide bond that
defines the conformations a polypeptide chain may assume. Within a single
protein different regions of the polypeptide chain may assume different
conformations determined by the primary sequence of the amino acids.
The α-Helix
The α-helix is a common secondary structure encountered in proteins of
the globular class. The formation of the α-helix is spontaneous and is
stabilized by H-bonding between amide nitrogens and carbonyl carbons of
peptide bonds spaced four residues apart. This orientation of H-bonding
produces a helical coiling of the peptide backbone such that the R-groups lie
on the exterior of the helix and perpendicular to its axis.
Typical α-Helix
Not all amino acids favor the formation of the (α-helix due to steric
constraints of the R-groups. Amino acids such as A, D, E, I, L and M favor the
formation of α-helices, whereas, G and P favor disruption of the helix. This is
particularly true for P since it is a pyrrolidine based imino acid (HN=) whose
structure significantly restricts movement about the peptide bond in which it is
present, thereby, interfering with extension of the helix. The disruption of the
helix is important as it introduces additional folding of the polypeptide
backbone to allow the formation of globular proteins.
β-Sheets
Whereas an α-helix is composed of a single linear array of helically
disposed amino acids, β-sheets are composed of 2 or more different regions
of stretches of at least 5-10 amino acids. The folding and alignment of
stretches of the polypeptide backbone aside one another to form β-sheets is
stabilized by H-bonding between amide nitrogens and carbonyl carbons.
However, the H-bonding residues are present in adjacently opposed stretches
of the polypetide backbone as opposed to a linearly contiguous region of the
backbone in the α-helix. β-sheets are said to be pleated. This is due to
positioning of the α-carbons of the peptide bond which alternates above and
below the plane of the sheet. β-sheets are either parallel or antiparallel. In
parallel sheets adjacent peptide chains proceed in the same direction (i.e. the
direction of N-terminal to C-terminal ends is the same), whereas, in
antiparallel sheets adjacent chains are aligned in opposite directions. β-sheets
can be depicted in ball and stick format or as ribbons in certain protein
formats.
Ball and Stick Representation of a βSheet
Ribbon Depiction of βSheet
Super-Secondary Structure
Some proteins contain an ordered organization of secondary structures
that form distinct functional domains or structural motifs. Examples include the
helix-turn-helix domain of bacterial proteins that regulate transcription and the
leucine zipper, helix-loop-helix and zinc finger domains of eukaryotic
transcriptional regulators. These domains are termed super-secondary
structures.
Tertiary Structure of Proteins
Tertiary structure refers to the complete three-dimensional structure of the
polypeptide units of a given protein. Included in this description is the spatial
relationship of different secondary structures to one another within a
polypeptide chain and how these secondary structures themselves fold into
the three-dimensional form of the protein. Secondary structures of proteins
often constitute distinct domains. Therefore, tertiary structure also describes
the relationship of different domains to one another within a protein. The
interactions of different domains is governed by several forces: These include
hydrogen bonding, hydrophobic interactions, electrostatic interactions and van
der Waals forces.
Forces Controlling Protein Structure
Hydrogen Bonding:
Polypeptides contain numerous proton donors and acceptors both in their
backbone and in the R-groups of the amino acids. The environment in which
proteins are found also contains the ample H-bond donors and acceptors of
the water molecule. H-bonding, therefore, occurs not only within and between
polypeptide chains but with the surrounding aqueous medium.
Hydrophobic Forces:
Proteins are composed of amino acids that contain either hydrophilic or
hydrophobic R-groups. It is the nature of the interaction of the different Rgroups with the aqueous environment that plays the major role in shaping
protein structure. The spontaneous folded state of globular proteins is a
reflection of a balance between the opposing energetics of H-bonding
between hydrophilic R-groups and the aqueous environment and the
repulsion from the aqueous environment by the hydrophobic R-groups. The
hydrophobicity of certain amino acid R-groups tends to drive them away from
the exterior of proteins and into the interior. This driving force restricts the
available conformations into which a protein may fold.
Electrostatic Forces:
Electrostatic forces are mainly of three types; charge-charge, charge-dipole
and dipole-dipole. Typical charge-charge interactions that favor protein folding
are those between oppositely charged R-groups such as K or R and D or E. A
substantial component of the energy involved in protein folding is chargedipole interactions. This refers to the interaction of ionized R-groups of amino
acids with the dipole of the water molecule. The slight dipole moment that
exist in the polar R-groups of amino acid also influences their interaction with
water. It is, therefore, understandable that the majority of the amino acids
found on the exterior surfaces of globular proteins contain charged or polar Rgroups.
van der Waals Forces:
There are both attractive and repulsive van der Waals forces that control
protein folding. Attractive van der Waals forces involve the interactions among
induced dipoles that arise from fluctuations in the charge densities that occur
between adjacent uncharged non-bonded atoms. Repulsive van der Waals
forces involve the interactions that occur when uncharged non-bonded atoms
come very close together but do not induce dipoles. The repulsion is the result
of the electron-electron repulsion that occurs as two clouds of electrons begin
to overlap.
Although van der Waals forces are extremely weak, relative to other forces
governing conformation, it is the huge number of such interactions that occur
in large protein molecules that make them significant to the folding of proteins.
Quaternary Structure
Many proteins contain 2 or more different polypeptide chains that are held
in association by the same non-covalent forces that stabilize the tertiary
structures of proteins. Proteins with multiple polypetide chains are oligomeric
proteins. The structure formed by monomer-monomer interaction in an
oligomeric protein is known as quaternary structure.
Oligomeric proteins can be composed of multiple identical polypeptide
chains or multiple distinct polypeptide chains. Proteins with identical subunits
are termed homo-oligomers. Proteins containing several distinct polypeptide
chains are termed hetero-oligomers.
Hemoglobin, the oxygen carrying protein of the blood, contains two α and
two β subunits arranged with a quaternary structure in the form, α 2β2.
Hemoglobin is, therefore, a hetero-oligomeric protein.
Structure of Hemoglobin
Complex Protein Structures
Proteins also are found to be covalently conjugated with carbohydrates.
These modifications occur following the synthesis (translation) of proteins and
are, therefore, termed post-translational modifications. These forms of
modification impart specialized functions upon the resultant proteins. Proteins
covalently associated with carbohydrates are termed glycoproteins.
Glycoproteins are of two classes, N-linked and O-linked, referring to the site of
covalent attachment of the sugar moieties. N-linked sugars are attached to
the amide nitrogen of the R-group of asparagine; O-linked sugars are
attached to the hydroxyl groups of either serine or threonine and occasionally
to the hydroxyl group of the modified amino acid, hydroxylysine.
There are extremely important glycoproteins found on the surface of
erythrocytes. It is the variability in the composition of the carbohydrate
portions of many glycoproteins and glycolipids of erythrocytes that determines
blood group specificities. There are at least 100 blood group determinants,
most of which are due to carbohydrate differences. The most common blood
groups, A, B, and O, are specified by the activity of specific gene products
whose activities are to incorporate distinct sugar groups onto RBC membrane
glycoshpingolipids as well as secreted glycoproteins.
Structural complexes involving protein associated with lipid via noncovalent
interactions are termed lipoproteins. The distinct roles of lipoproteins are
described on the linked page. Their major function in the body is to aid in the
storage transport of lipid and cholesterol.
Clinical Significances
This discussion is not intended to be a complete review of all disorders that
result from defects in protein structure and function. Visit the Inborn Errors
page for a more complete listing of diseases related to abnormal proteins and
also click on the links to the specific examples below for more information.
The substitution of a hydrophobic amino acid (V) for an acidic amino acid
(E) in the β-chain of hemoglobin results in sickle cell anemia (HbS). This
change of a single amino acid alters the structure of hemoglobin molecules in
such a way that the deoxygenated proteins polymerize and precipitate within
the erythrocyte, leading to their characteristic sickle shape.
Collagens are the most abundant proteins in the body. Alterations in
collagen structure arising from abnormal genes or abnormal processing of
collagen proteins results in numerous diseases, including Larsen syndrome,
scurvy, osteogenesis imperfecta and Ehlers-Danlos syndrome.
Ehlers-Danlos syndrome is actually the name associated with at least ten
distinct disorders that are biochemically and clinically distinct yet all manifest
structural weakness in connective tissue as a result of defective collagen
structure. Osteogenesis imperfecta also encompasses more than one
disorder. At least four biochemically and clinically distinguishable maladies
have been identified as osteogenesis imperfecta, all of which are
characterized by multiple fractures and resultant bone deformities. Marfan
syndrome manifests itself as a disorder of the connective tissue and was
originally believed to be the result of abnormal collagens. However, recent
evidence has shown that Marfan syndrome results from mutations in the
extracellular protein, fibrillin, which is an integral constituent of the noncollagenous microfibrils of the extracellular matrix.
Several forms of familial hypercholesterolemia are the result of genetic
defects in the gene encoding the receptor for low-density lipoprotein (LDL).
These defects result in the synthesis of abnormal LDL receptors that are
incapable of binding to LDLs, or that bind LDLs but the receptor/LDL
complexes are not properly internalized and degraded. The outcome is an
elevation in serum cholesterol levels and increased propensity toward the
development of atherosclerosis.
A number of proteins can contribute to cellular transformation and
carcinogenesis when their basic structure is disrupted by mutations in their
genes. These genes are termed proto-oncogenes. For some of these
proteins, all that is required to convert them to the oncogenic form is a single
amino acid substitution. The cellular gene, RAS, is observed to sustain single
amino acid substitutions at positions 12 or 61 with high frequency in colon
carcinomas. Mutations in RAS are most frequently observed genetic
alterations in colon cancer.
Myoglobin
Myoglobin and hemoglobin are hemeproteins whose physiological
importance is principally related to their ability to bind molecular oxygen.
Myoglobin is a monomeric heme protein found mainly in muscle tissue where
it serves as an intracellular storage site for oxygen. During periods of oxygen
deprivation oxymyoglobin releases its bound oxygen which is then used for
metabolic purposes.
The tertiary structure of myoglobin is that of a typical water soluble globular
protein. Its secondary structure is unusual in that it contains a very high
proportion (75%) of α-helical secondary structure. A myoglobin polypeptide is
comprised of 8 separate right handed α-helices, designated A through H, that
are connected by short non helical regions. Amino acid R-groups packed into
the interior of the molecule are predominantly hydrophobic in character while
those exposed on the surface of the molecule are generally hydrophilic, thus
making the molecule relatively water soluble.
Structure of Myoglobin with Heme
Each myoglobin molecule contains one heme prosthetic group inserted into
a hydrophobic cleft in the protein. Each heme residue contains one central
coordinately bound iron atom that is normally in the Fe 2+, or ferrous, oxidation
state. The oxygen carried by hemeproteins is bound directly to the ferrous iron
atom of the heme prosthetic group. Oxidation of the iron to the Fe3+, ferric,
oxidation state renders the molecule incapable of normal oxygen binding.
Hydrophobic interactions between the tetrapyrrole ring and hydrophobic
amino acid R groups on the interior of the cleft in the protein strongly stabilize
the heme protein conjugate. In addition a nitrogen atom from a histidine R
group located above the plane of the heme ring is coordinated with the iron
atom further stabilizing the interaction between the heme and the protein. In
oxymyoglobin the remaining bonding site on the iron atom (the 6th coordinate
position) is occupied by the oxygen, whose binding is stabilized by a second
histidine residue.
Carbon monoxide also binds coordinately to heme iron atoms in a manner
similar to that of oxygen, but the binding of carbon monoxide to heme is much
stronger than that of oxygen. The preferential binding of carbon monoxide to
heme iron is largely responsible for the asphyxiation that results from carbon
monoxide poisoning.
Hemoglobin
Adult hemoglobin is a [α(2):β(2)] tetrameric hemeprotein found in
erythrocytes where it is responsible for binding oxygen in the lung and
transporting the bound oxygen throughout the body where it is used in aerobic
metabolic pathways.
Structure of Hemoglobin
For a description of the different types of hemoglobin tetramers see the
section below on Hemoglobin Genes. Each subunit of a hemoglobin tetramer
has a heme prosthetic group identical to that described for myoglobin. The
common peptide subunits are designated α, β, γ and δ which are arranged
into the most commonly occurring functional hemoglobins.
Although the secondary and tertiary structure of various hemoglobin
subunits are similar, reflecting extensive homology in amino acid composition,
the variations in amino acid composition that do exist impart marked
differences in hemoglobin's oxygen carrying properties. In addition, the
quaternary structure of hemoglobin leads to physiologically important
allosteric interactions between the subunits, a property lacking in monomeric
myoglobin which is otherwise very similar to the α-subunit of hemoglobin.
Comparison of the oxygen binding properties of myoglobin and hemoglobin
illustrate the allosteric properties of hemoglobin that results from its
quaternary structure and differentiate hemoglobin's oxygen binding properties
from that of myoglobin. The curve of oxygen binding to hemoglobin is
sigmoidal typical of allosteric proteins in which the substrate, in this case
oxygen, is a positive homotropic effector. When oxygen binds to the first
subunit of deoxyhemoglobin it increases the affinity of the remaining subunits
for oxygen. As additional oxygen is bound to the second and third subunits
oxygen binding is further, incrementally, strengthened, so that at the oxygen
tension in lung alveoli, hemoglobin is fully saturated with oxygen. As
oxyhemoglobin circulates to deoxygenated tissue, oxygen is incrementally
unloaded and the affinity of hemoglobin for oxygen is reduced. Thus at the
lowest oxygen tensions found in very active tissues the binding affinity of
hemoglobin for oxygen is very low allowing maximal delivery of oxygen to the
tissue. In contrast the oxygen binding curve for myoglobin is hyperbolic in
character indicating the absence of allosteric interactions in this process.
The allosteric oxygen binding properties of hemoglobin arise directly from
the interaction of oxygen with the iron atom of the heme prosthetic groups and
the resultant effects of these interactions on the quaternary structure of the
protein. When oxygen binds to an iron atom of deoxyhemoglobin it pulls the
iron atom into the plane of the heme. Since the iron is also bound to histidine
F8, this residue is also pulled toward the plane of the heme ring. The
conformational change at histidine F8 is transmitted throughout the peptide
backbone resulting in a significant change in tertiary structure of the entire
subunit. Conformational changes at the subunit surface lead to a new set of
binding interactions between adjacent subunits. The latter changes include
disruption of salt bridges and formation of new hydrogen bonds and new
hydrophobic interactions, all of which contribute to the new quaternary
structure.
The latter changes in subunit interaction are transmitted, from the surface,
to the heme binding pocket of a second deoxy subunit and result in easier
access of oxygen to the iron atom of the second heme and thus a greater
affinity of the hemoglobin molecule for a second oxygen molecule. The tertiary
configuration of low affinity, deoxygenated hemoglobin (Hb) is known as the
taut (T) state. Conversely, the quaternary structure of the fully oxygenated
high affinity form of hemoglobin (HbO2) is known as the relaxed (R) state.
In the context of the affinity of hemoglobin for oxygen there are four primary
regulators, each of which has a negative impact. These are CO2, hydrogen
ion (H+), chloride ion (Cl–), and 2,3-bisphosphoglycerate (2,3BPG, or also just
BPG). Some older texts abbreviate 2,3BPG as DPB. Although they can
influence O2 binding independent of each other, CO2, H+ and Cl– primarily
function as a consequence of each other on the affinity of hemoglobin for O 2.
We shall consider the transport of O2 from the lungs to the tissues first.
In the high O2 environment (high pO2) of the lungs there is sufficient O2 to
overcome the inhibitory nature of the T state. During the O2 binding-induced
alteration from the T form to the R form several amino acid side groups on the
surface of hemoglobin subunits will dissociate protons as depicted in the
equation below. This proton dissociation plays an important role in the
expiration of the CO2 that arrives from the tissues (see below). However,
because of the high pO2, the pH of the blood in the lungs (≈7.4 – 7.5) is not
sufficiently low enough to exert a negative influence on hemoglobin binding
O2. When the oxyhemoglobin reaches the tissues the pO 2 is sufficiently low,
as well as the pH (≈7.2), that the T state is favored and the O2 released.
4O2 + Hb <——> nH+ + Hb(O2)4
If we now consider what happens in the tissues, it is possible to see how
CO2, H+, and Cl– exert their negative effects on hemoglobin binding O2.
Metabolizing cells produce CO2 which diffuses into the blood and enters the
circulating red blood cells (RBCs). Within RBCs the CO2 is rapidly converted
to carbonic acid through the action of carbonic anhydrase as shown in the
equation below:
CO2 + H2O ——> H2CO3 ——> H+ + HCO3–
The bicarbonate ion produced in this dissociation reaction diffuses out of
the RBC and is carried in the blood to the lungs. This effective CO 2 transport
process is referred to as isohydric transport. Approximately 80% of the CO 2
produced in metabolizing cells is transported to the lungs in this way. A small
percentage of CO2 is transported in the blood as a dissolved gas. In the
tissues, the H+ dissociated from carbonic acid is buffered by hemoglobin
which exerts a negative influence on O2 binding forcing release to the tissues.
As indicated above, within the lungs the high pO2 allows for effective O2
binding by hemoglobin leading to the T to R state transition and the release of
protons. The protons combine with the bicarbonate that arrived from the
tissues forming carbonic acid which then enters the RBCs. Through a reversal
of the carbonic anhydrase reaction, CO2 and H2O are produced. The CO2
diffuses out of the blood, into the lung alveoli and is released on expiration.
In addition to isohydric transport, as much as 15% of CO 2 is transported to
the lungs bound to N-terminal amino groups of the T form of hemoglobin. This
reaction, depicted below, forms what is called carbamino-hemoglobin. As
indicated this reaction also produces H+, thereby lowering the pH in tissues
where the CO2 concentration is high. The formation of H+ leads to release of
the bound O2 to the surrounding tissues. Within the lungs, the high O2 content
results in O2 binding to hemoglobin with the concomitant release of H +. The
released protons then promote the dissociation of the carbamino to form CO2
which is then released with expiration.
CO2 + Hb-NH2 <——> H+ + Hb-NH-COO–
As the above discussion demonstrates, the conformation of hemoglobin
and its oxygen binding are sensitive to hydrogen ion concentration. These
effects of hydrogen ion concentration are responsible for the well known Bohr
effect in which increases in hydrogen ion concentration decrease the amount
of oxygen bound by hemoglobin at any oxygen concentration (partial
pressure). Coupled to the diffusion of bicarbonate out of RBCs in the tissues
there must be ion movement into the RBCs to maintain electrical neutrality.
This is the role of Cl- and is referred to as the chloride shift. In this way, Cl–
plays an important role in bicarbonate production and diffusion and thus also
negatively influences O2 binding to hemoglobin.
Representation of the transport of CO2 from the tissues to the blood with
delivery of O2 to the tissues. The opposite process occurs when O2 is taken
up from the alveoli of the lungs and the CO2 is expelled. All of the processes
of the transport of CO2 and O2 are not shown such as the formation and
ionization of carbonic acid in the plasma. The latter is a major mechanism for
the transport of CO2 to the lungs, i.e. in the plasma as HCO3–. The H+
produced in the plasma by the ionization of carbonic acid is buffered by
phosphate (HPO42–) and by proteins. Additionally, some 15% of the CO 2 is
transported from the tissues to the lungs as hemoglobin carbamate.
Role of 2,3-bisphosphoglycerate (2,3-BPG)
The compound 2,3-bisphosphoglycerate (2,3-BPG), derived from the
glycolytic intermediate 1,3-bisphosphoglycerate, is a potent allosteric effector
on the oxygen binding properties of hemoglobin. The pathway to 2,3BPG
synthesis is diagrammed in the figure below.
The pathway for 2,3-bisphosphoglycerate (2,3-BPG) synthesis within
erythrocytes. Synthesis of 2,3-BPG represents a major reaction pathway for
the consumption of glucose in erythrocytes. The synthesis of 2,3-BPG in
erythrocytes is critical for controlling hemoglobin affinity for oxygen. Note that
when glucose is oxidized by this pathway the erythrocyte loses the ability to
gain 2 moles of ATP from glycolytic oxidation of 1,3-BPG to 3phosphoglycerate via the phosphoglycerate kinase reaction.
In the deoxygenated T conformer, a cavity capable of binding 2,3-BPG
forms in the center of the molecule. 2,3-BPG can occupy this cavity stabilizing
the T state. Conversely, when 2,3-BPG is not available, or not bound in the
central cavity, Hb can be converted to HbO2 more readily. Thus, like
increased hydrogen ion concentration, increased 2,3-BPG concentration
favors conversion of R form Hb to T form Hb and decreases the amount of
oxygen bound by Hb at any oxygen concentration. Hemoglobin molecules
differing in subunit composition are known to have different 2,3-BPG binding
properties with correspondingly different allosteric responses to 2,3-BPG. For
example, HbF (the fetal form of hemoglobin) binds 2,3-BPG much less avidly
than HbA (the adult form of hemoglobin) with the result that HbF in fetuses of
pregnant women binds oxygen with greater affinity than the mothers HbA,
thus giving the fetus preferential access to oxygen carried by the mothers
circulatory system.
The Hemoglobin Genes
The α- and β-globin proteins contained in functional hemoglobin tetramers
are derived from gene clusters. The α-globin genes are on chromosome 16
and the β-globin genes are on chromosome 11. Both gene clusters contain
not only the major adult genes, α and β, but other expressed sequences that
are utilized at different stages of development. The orientation of the genes in
both clusters is in the same 5' to 3' direction with the earliest expressed genes
at the 5' end of both clusters. In addition to functional genes, both clusters
contain non-functional pseudogenes.
Hemoglobin synthesis begins in the first few weeks of embryonic
development within the yolk sac. The major hemoglobin at this stage of
development is a tetramer composed of 2 zeta (ζ) chains encoded within the α
cluster and 2 epsilon (ε) chains from the β cluster. By 6-8 weeks of gestation
the expression of this version of hemoglobin declines dramatically coinciding
with the change in hemoglobin synthesis from the yolk sac to the liver.
Expression from the α cluster consists of identical proteins from the α1 and α2
genes. Expression from these genes in the α cluster remains on throughout
life.
Within the β-globin cluster there is an additional set of genes, the fetal βglobin genes identified as the gamma (γ) genes. The 2 fetal genes called Gγ
and Aγ, the derivation of which stems from the single amino acid difference
between the 2 fetal genes: glycine in Gγ and alanine in Aγ at position 136.
These fetal γ genes are expressed as the embryonic genes are turned off.
Shortly before birth there is a smooth switch from fetal γ-globin gene
expression to adult β-globin gene expression. The switch from fetal γ- to adult
β-globin does not directly coincide with the switch from hepatic synthesis to
bone marrow synthesis since at birth it can be shown that both γ and β
synthesis is occurring in the marrow.
Given the pattern of globin gene activity throughout fetal development and
in the adult the composition of the hemoglobin tetramers is of course distinct.
Fetal hemoglobin is identified as HbF and includes both α2Gγ2 and α2Aγ2.
Fetal hemoglobin has a slightly higher affinity for oxygen than does adult
hemoglobin. This allows the fetus to extract oxygen more efficiently from the
maternal circulation. In adults the major hemoglobin is identified as HbA
(more commonly HbA1) and is a tetramer of 2 α and 2 β chains as indicated
earlier. A minor adult hemoglobin, identified as HbA 2, is a tetramer of 2 α
chains and 2 δ chains. The δ gene is expressed with a timing similar to the β
gene but because the promoter has acquired a number of mutations its'
efficiency of transcription is reduced.
The overall hemoglobin composition in a normal adult is approximately
97.5% HbA1, 2% HbA2 and 0.5% HbF.
Hemoglobinopathies
A large number of mutations have been described in the globin genes.
These mutations can be divided into two distinct types: those that cause
qualitative abnormalities (e.g. sickle cell anemia) and those that cause
quantitative abnormalities (the thalassemias). Taken together these
disorders are referred to as the hemoglobinopathies. A third group of
hemoglobin disorders include those diseases in which there is a persistence
of fetal hemoglobin expression. These latter diseases are known collectively
as hereditary persistence of fetal hemoglobin (HPFH).
Of the mutations leading to qualitative alterations in hemoglobin, the
missense mutation in the β-globin gene that causes sickle cell anemia is the
most common. The mutation causing sickle cell anemia is a single nucleotide
substitution (A to T) in the codon for amino acid 6. The change converts a
glutamic acid codon (GAG) to a valine codon (GTG). The form of hemoglobin
in persons with sickle cell anemia is referred to as HbS.
The underlying problem in sickle cell anemia is that the valine for glutamic
acid substitution results in hemoglobin tetramers that aggregate into arrays
upon deoxygenation in the tissues. This aggregation leads to deformation of
the red blood cell making it relatively inflexible and unable to traverse the
capillary beds. Repeated cycles of oxygenation and deoxygenation lead to
irreversible sickling. The end result is clogging of the fine capillaries. Because
bones are particularly affected by the reduced blood flow, frequent and severe
bone pain results. This is the typical symptom during a sickle cell "crisis".
Long term the recurrent clogging of the capillary beds leads to damage to the
internal organs, in particular the kidneys, heart and lungs. The continual
destruction of the sickled red blood cells leads to chronic anemia and
episodes of hyperbilirubinemia.
An additional relatively common mutation at codon 6 is the conversion to a
lysine codon (AAG) which results in the generation of HbC.
Electrophoresis of hemoglobin proteins from individuals suspected of
having sickle cell anemia (or several other types of hemoglobin disorders) is
an effective diagnostic tool because the variant hemoglobins have different
charges. An example of this technique is shown in the Figure below.
Pattern
of
hemoglobin
electrophoresis from several different
individuals. Lanes 1 and 5 are
hemoglobin standards. Lane 2 is a
normal adult. Lane 3 is a normal
neonate. Lane 4 is a homozygous
HbS individual. Lanes 6 and 8 are
heterozygous sickle individuals. Lane
7 is a SC disease individual.
Another effective tool to identify the genotype of individuals suspected of
having sickle cell disease as well as for prenatal diagnosis is to either carry
out RFLP mapping or to use PCR. An example of the use of these tools can
be seen in the Molecular Tools of Medicine page.
In addition to the missense mutations that lead to HbS and HbC, a number
of frameshift mutations leading to qualitative abnormalities in hemoglobin
have been identified. A 2-nucleotide insertion between codons 144 and 145 in
the β-globin gene results in the generation of hemoglobin Cranston. The
insertion, which is near the C-terminus of the β-globin protein, results in the
normal stop codon being out of frame and synthesis proceeding into the 3'untranslated region to a fortuitous stop codon. The result is a β-globin protein
of 157 amino acids.
In the hemoglobin Constant Spring variant, a mutation in the α-globin
gene converts the stop codon (UAA) to a glutamine codon (CAA) so that the
protein ends up being 31 amino acids longer than normal. The resultant αglobin protein in hemoglobin Constant Spring is not only qualitatively altered
but because it is unstable it is a quantitative abnormality as well.
Because the globin gene loci contain clusters of similar genes there is the
potential for unequal cross-over between the sister chromatids during meiosis.
The generation of hemoglobin Gun Hill and Lepore hemoglobins are both
the result of unequal cross over events. Hemoglobin Gun Hill is the result of a
deletion of 15 nucleotides caused by unequal cross over between codons 91–
94 of one β-globin gene and codons 96–98 of the other. Generation of Lepore
hemoglobins results from unequal cross over between the δ-globin and βglobin genes. The resultant hybrid δβ gene is called Lepore and the βδ hybrid
gene is called anti-Lepore. As indicated earlier, the promoter of the δ-globin
gene is inefficient so the consequences of this unequal cross over event are
both qualitative and quantitative.
The thalassemias are the result of abnormalities in hemoglobin synthesis
and affect both clusters. Deficiencies in β-globin synthesis result in the βthalassemias and deficiencies in α-globin synthesis result in the αthalassemias. The term thalassemia is derived from the Greek thalassa
meaning "sea" and was applied to these disorders because of the high
frequency of their occurrence in individuals living around the Mediterranean
Sea.
In normal individuals an equal amount of both α- and β-globin proteins are
made allowing them to combine stoichiometrically to form the correct
hemoglobin tetramers. In the α-thalassemias normal amounts of β-globin are
made. The β-globin proteins are capable of forming homotetramers (β 4) and
these tetramers are called hemoglobin H, (HbH). An excess of HbH in red
blood cells leads to the formation if inclusion bodies commonly seen in
patients with α-thalassemia. In addition, the HbH tetramers have a markedly
reduced oxygen carrying capacity. In β-thalassemia, where the β-globins are
deficient, the α-globins are in excess and will form α-globin homotetramers.
The α-globin homotetramers are extremely insoluble which leads to premature
red cell destruction in the bone marrow and spleen.
With the α-thalassemias the level of α-globin production can range from
none to very nearly normal levels. This is due in part to the fact that there are
2 identical α-globin genes on chromosome 16. Thus, the α-thalassemias
involve inactivation of 1 to all 4 α-globin genes. If 3 of the 4 α-globin genes are
functional, individuals are completely asymptomatic. This situation is identified
as the "silent carrier" state or sometimes as α-thalassemia 2. Genotypically
this situation is designated αα/α– (where the dash indicates a non-functional
gene) or α–/αα. If 2 of the 4 genes are inactivated individuals are designated
as α-thalassemia trait or as α-thalassemia 1. Genotypically this situation is
designated αα/– –. In individuals of African descent with α-thalassemia 1, the
disorder usually results from the inactivation of 1 α-globin gene on each
chromosome and is designated α–/α–. This means that these individuals are
homozygous for the α-thalassemia 2 chromosome. The phenotype of αthalassemia 1 is relatively benign. The mean red cell volume (designated
MCV in clinical tests) is reduced in α-thalassemia 1 but individuals are
generally asymptomatic. The clinical situation becomes more severe if only 1
of the 4 α-globin genes is functional. Because of the dramatic reduction in αglobin chain production in this latter situation, a high level of β 4 tetramer is
present. clinically this is referred to as hemoglobin H disease. Afflicted
individuals have moderate to marked anemia and their MCV is quite low, but
the disease is not fatal. The most severe situation results when no α-globin
chains are made (genotypically designated – –/– –). This leads to prenatal
lethality or early neonatal death. The predominant fetal hemoglobin in afflicted
individuals is a tetramer of γ-chains and is referred to as hemoglobin Bart's.
This hemoglobin has essentially no oxygen carrying capacity resulting in
oxygen starvation in the fetal tissues. Heart failure results as the heart tries to
pump the unoxygenated blood to oxygen starved tissues leading to marked
edema. This latter situation is called hydrops fetalis.
A large number of mutations have been identified leading to decreased or
absent production of β-globin chains resulting in the β-thalassemias. In the
most severe situation mutations in both the maternal and paternal β-globin
genes leads to loss of normal amounts of β-globin protein. A complete lack of
HbA is denoted as β0-thalassemia. If one or the other mutations allows
production of a small amount of functional β-globin then the disorder is
denoted as β+-thalassemia.
Both β0- and β+-thalassemias are referred to as thalassemia major, also
called Cooley's anemia after Dr. Thomas Cooley who first described the
disorder. Afflicted individuals suffer from severe anemia beginning in the first
year of life leading to the need for blood transfusions. As a consequence of
the anemia the bone marrow dramatically increases its' effort at blood
production. The cortex of the bone becomes thinned leading to pathologic
fracturing and distortion of the bones in the face and skull. In addition, there is
marked hepatosplenomegaly as the liver and spleen act as additional sites of
blood production. Without intervention these individuals will die within the
decade of life. As indicated, β-thalassemia major patient require blood
transfusions, however, in the long term these transfusions lead to the
accumulation of iron in the organs, particularly the heart, liver and pancreas.
Organ failure ensues with death in the teens to early twenties. Iron chelation
therapies appear to improve the outlook for β-thalassemia major patients but
this requires continuous infusion of the chelating agent.
Individuals heterozygous for β-thalassemia have what is termed
thalassemia minor. Afflicted individuals harbor one normal β-globin gene and
one that harbors a mutation leading to production of reduced or no β-globin.
Individuals that do not make any functional β-globin protein from 1 gene are
termed βo heterozygotes. If β-globin production is reduced at one locus the
individuals are termed β+ heterozygotes. Thalassemia minor individuals are
generally asymptomatic.
The term thalassemia intermedia is used to designate individuals with
significant anemia and who are symptomatic but unlike thalassemia major do
not require transfusions. This syndrome results in individuals where both βglobin genes express reduced amounts of protein or where one gene makes
none and the other makes a mildly reduced amount. A person who is a
compound heterozygote with α-thalassemia and β+-thalassemia will also
manifest as thalassemia intermedia.
The primary cause of α-thalassemias is deletion, whereas, for βthalassemias the mutations are more subtle. In β-thalassemias, point
mutations in the promoter, mutations in the translational initiation codon, a
point mutation in the polyadenylation signal and an array of mutations leading
to splicing abnormalities have been characterized.
An interesting and common (up to 30% of persons from Southeast Asia)
hemoglobinopathy that has both quantitative and qualitative characteristics is
caused by the synthesis of hemoglobin E. Hemoglobin E arises due to a point
mutation in codon 26 that changes glutamic acid (GAG) to lysine (AAG).
Individulas with this mutation make only around 60% of the normal amount of
β-globin protein. The reason for this is that the mutation creates a cryptic
splice site such that 40% of the hemoglobin E mRNA is shorter by 16
nucleotides and does not give rise to detectable β-globin protein.
There are some individuals in whom the developmental timing of globin
production is altered as a consequence of mutation. Persons with hereditary
persistence of fetal hemoglobin, HPFH continue to make HbF as adults.
Because the syndrome is benign most individuals do not even know they
carry a hemoglobin abnormality. Many HPFH individuals harbor large
deletions of the δ- and β-coding region of the cluster. There is no deletion of
the fetal globin genes and by an as yet uncharacterized mechanism
expression of these genes persists in adulthood.
As discussed above functional hemoglobin is a heterotetramer. Mutations
in either the α-globin or the β-globin genes lead to quantitative and qualitative
abnormalities in hemoglobin. Therefore, it should not be surprising that
complex compound heterozygosities can result in offspring of individuals
harboring different mutations.
Source: medical biochemistry web page